Clinical Features, Tumor Biology, and Prognosis Associated with MYC

Modern Pathology (2015) 28, 1555–1573

Clinical features, tumor biology, and prognosis associated with MYC rearrangement and Myc overexpression in diffuse large B-cell lymphoma patients treated with rituximab-CHOP Zijun Y Xu-Monette1, Bouthaina S Dabaja2, Xiaoxiao Wang1, Meifeng Tu1, Ganiraju C Manyam3, Alexander Tzankov4, Yi Xia1, Li Zhang3, Ruifang Sun1, Carlo Visco5, Karen Dybkaer6, Lihui Yin1, April Chiu7, Attilio Orazi8, Youli Zu9, Govind Bhagat10, Kristy L Richards11, Eric D Hsi12, William WL Choi13, J Han van Krieken14, Jooryung Huh15, Maurilio Ponzoni16, Andrés JM Ferreri16, Michael B Møller17, Ben M Parsons18, Xiaoying Zhao19, Jane N Winter20, Miguel A Piris21, Timothy J McDonnell1, Roberto N Miranda1, Yong Li22, L Jeffrey Medeiros1 and Ken H Young1,23

1Department of Hematopathology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; 2Department of Radiation Oncology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; 3Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA; 4University Hospital, Basel, Switzerland; 5San Bortolo Hospital, Vicenza, Italy; 6Aalborg University Hospital, Aalborg, Denmark; 7Memorial Sloan-Kettering Cancer Center, New York, NY, USA; 8Weill Medical College of Cornell University, New York, NY, USA; 9The Methodist Hospital, Houston, TX, USA; 10Columbia University Medical Center and New York Presbyterian Hospital, New York, NY, USA; 11University of North Carolina School of Medicine, Chapel Hill, NC, USA; 12Cleveland Clinic, Cleveland, OH, USA; 13University of Hong Kong Li Ka Shing Faculty of Medicine, Hong Kong, China; 14Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands; 15Asan Medical Center, Ulsan University College of Medicine, Seoul, South Korea; 16San Raffaele H Scientific Institute, Milan, Italy; 17Odense University Hospital, Odense, Denmark; 18Gundersen Lutheran Health System, La Crosse, WI, USA; 19Zhejiang University School of Medicine, Second University Hospital, Hangzhou, China; 20Feinberg School of Medicine, Northwestern University, Chicago, IL, USA; 21Hospital Universitario Marqués de Valdecilla, Santander, Spain; 22Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA and 23The University of Texas School of Medicine, Graduate School of Biomedical Sciences, Houston, TX, USA

MYC dysregulation, including MYC gene rearrangement and Myc protein overexpression, is of increasing clinical importance in diffuse large B-cell lymphoma (DLBCL). However, the roles of MYC and the relative importance of rearrangement vs overexpression remain to be refined. Gaining knowledge about the tumor biology associated with MYC dysregulation is important to understand the roles of MYC and MYC-associated biology in lymphomagenesis. In this study, we determined MYC rearrangement status (n = 344) and Myc expression (n = 535) in a well-characterized DLBCL cohort, individually assessed the clinical and pathobiological features of patients with MYC rearrangement and Myc protein overexpression, and analyzed the prognosis and gene expression profiling signatures associated with these MYC abnormalities in germinal center B-cell-like and activated B-cell-like DLBCL. Our results showed that the prognostic importance of MYC rearrangement vs Myc overexpression is significantly different in germinal center B-cell-like vs activated B-cell-like DLBCL. In germinal center B-cell-like DLBCL, MYC-rearranged germinal center B-cell-like DLBCL patients with Myc overexpression significantly contributed to the clinical, biological, and prognostic characteristics of the overall

Correspondence: Dr KH Young, MD, Department of Hematopathology, The University of Texas MD Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA. E-mail: [email protected] Received 10 July 2015; revised 16 August 2015; accepted 29 August 2015; published online 6 November 2015 www.modernpathology.org MYC-associated biology and prognosis in DLBCL 1556 ZY Xu-Monette et al

Myc-overexpressing germinal center B-cell-like DLBCL group. In contrast, in activated B-cell-like DLBCL, the occurrence, clinical and biological features, and prognosis of Myc overexpression were independent of MYC rearrangement. High Myc levels and Myc-independent mechanisms, either tumor cell intrinsic or related to tumor microenvironment, conferred significantly worse survival to MYC-rearranged germinal center B-cell-like DLBCL patients, even among MychighBcl-2high DLBCL patients. This study provides new insight into the tumor biology and prognostic effects associated with MYC dysregulation and suggest that detection of both MYC translocations and evaluation of Myc and Bcl-2 expression is necessary to predict the prognosis of DLBCL patients. Modern Pathology (2015) 28, 1555–1573; doi:10.1038/modpathol.2015.118; published online 6 November 2015

MYC is a proto-oncogene that encodes the Myc Possible molecular mechanisms underlying the protein, which is critical for cell proliferation, growth, inconsistent clinical results may include presence or metabolism, differentiation, apoptosis, and immune absence of other genetic abnormalities and onco- – responses. In mouse models, Myc inactivation induces genic pathways,29 31 as well as another aspect of sustained tumor regression via both tumor cell- Myc function: promoting apoptosis.32 Moreover, intrinsic and host-dependent mechanisms.1 The selec- researchers have recently reported findings that tive small-molecule bromodomain inhibitor JQ1 has a Myc is a universal amplifier of 10–15% in human potent antiproliferative effect that was associated with genome, suggesting that Myc function is nonspecific the effective downregulation of MYC and Myc target and that the consequences of MYC activities are genes2 rendering this agent to have great therapeutic affected by pre-existing molecular programs in the potential.3 tumor cells.33,34 Therefore, tumor biology associated The chromosomal rearrangement or translocation with MYC/Myc (designated MYC herein) abnormal- involving MYC and other genes (most commonly the ities may have important roles in the observed immunoglobulin heavy-chain gene (IGH) locus) adverse prognosis. leads to Myc overexpression and occurs in ~ 10% Taken together, both MYC gene rearrangement and of diffuse large B-cell lymphomas (DLBCLs).4–8 Myc protein overexpression have been correlated DLBCL is the most common type of non-Hodgkin's with significantly adverse prognosis in DLBCL. lymphoma, and among MYC-rearranged aggressive However, how much of these two biomarkers over- lymphomas, DLBCL is the entity that clinicians most lap and differ, how much their associated tumor commonly encounter.9,10 Several studies have biology affects the prognostic effects, and whether reported that MYC translocations independently the MYC functional role is molecular context- predicted significantly poor survival in DLBCL dependent are not very clear. In this study, we patients.11–16 However, other studies found incon- compared the occurrence and clinicopathologic sistent results17–19 or limitations of its prognostic features of patients stratified by MYC rearrangement significance.20,21 and Myc expression status, and analyzed the The clinical significance of Myc overexpression in differential prognosis and gene expression profiling DLBCL has also been the source of much attention associated with these MYC abnormalities in a well- characterized DLBCL cohort to assess the utility of and controversy. Several groups including ours have these two genetic and protein biomarkers and found that DLBCL with high Myc protein expression explore the prognostic determinants. This study is detected by immunohistochemistry had inferior – important for achieving the goal of precision medi- survival.5,13,22 24 Furthermore, the poor prognosis cine in DLBCL. associated with Myc overexpression was contributed by cases with Myc/Bcl-2 coexpression—‘double- positive lymphoma’ (DPL)—which account for 18– 44% of DLBCLs.13,20,23–26 However, one study Patients and methods showed that the prognostic value of double-positive lymphoma was lost in younger DLBCL patients with Patients poor prognosis.27 Inconsistent results have been The study cohort consisted of 539 R-CHOP-treated reported regarding whether the prognostic signifi- patients with de novo DLBCL from the International cance of Myc or Myc/Bcl2 protein expression DLBCL Rituximab-CHOP Consortium Program, depends on MYC or MYC/BCL2 gene rearrangement including 466 cases from the training set of a status or not.8,13,20 Other issues include whether previous study,24 and additional 73 cases with either Myc/Bcl2 immunohistochemistry is robust and Myc immunohistochemistry or MYC gene rearrange- reproducible,28 and that immunohistochemistry cut- ment status determined. The diagnostic criteria, off values have varied among different study review process, and eligibility and exclusion criteria groups,13,20,23–26 which may affect the specificity of have been described previously.35,36 The cell-of- this combined biomarker for poorer prognosis. origin classification as either the germinal center

Modern Pathology (2015) 28, 1555–1573 MYC-associated biology and prognosis in DLBCL ZY Xu-Monette et al 1557

B-cell-like (GCB) or activated B-cell-like (ABC) Statistical Analysis subtype was determined using gene expression profiling and/or immunohistochemistry for CD10, The clinical and pathobiological features of DLBCL patients at the time of presentation were compared BCL6, GCET-1, FOXP1, and MUM1 using the Visco- χ2 Young and/or Choi algorithms as described using the test. The mean expression levels of previously.24,35,36 Totally, 276 cases were classified biomarkers between DLBCL groups with or without as GCB, 259 cases were classified as ABC, and 4 MYC abnormalities were also compared by unpaired cases were unclassifiable. All patients underwent t-test. Overall survival (OS) was calculated from the standard R-CHOP or R-CHOP-like therapy, and the time of diagnosis to last follow-up or death from any median follow-up time was 45 months (range, cause. Progression-free survival (PFS) was calculated 30–176.1 months). This study was conducted in from the time of diagnosis to disease progression, accordance with the Declaration of Helsinki and was disease relapse, or death from any cause. Patients approved as being of minimal to no risk or as exempt who were alive and disease progression-free at last follow-up were censored. Survival analysis was by the institutional review boards of all participating – centers, including The University of Texas MD performed using the Kaplan Meier method with Anderson Cancer Center. the Prism 5 program (GraphPad Software, San Diego, CA, USA), and differences in survival were compared using the log-rank (Mantel–Cox) test. Multi- Fluorescence In Situ Hybridization, variate survival analysis was performed using a Cox Immunohistochemistry, and Gene Sequencing proportional hazards regression model with the SPSS software program (version 19.0; IBM Corpora- MYC translocation was detected by fluorescence tion, Armonk, NY, USA). All differences with in situ hybridization using two probes (a locus- P ≤ 0.05 were considered statistically significant. specific identifier IGH/MYC/CEP8 tri-color dual- fusion probe and a locus-specific identifier MYC dual-color break-apart probe) (n = 344). Myc expres- Results sion was assessed by immunohistochemistry using tissue microarray sections and a monoclonal anti-(c) Myc Expression in Germinal Center B-Cell-Like and MYC antibody, clone Y69 (Epitomics, Burlingame, Activated-B-Cell-Like Subtypes of DLBCL CA, USA) (n = 535). The experimental techniques We observed variable levels of nuclear Myc expres- and scoring processes have been described sion in DLBCL (n = 535) (Figures 1a–c). The mean previously.15,21,24 expression level of Myc in ABC-DLBCL was signi- Evaluation of other biomarker expression by ficantly higher compared with that in GCB-DLBCL immunohistochemistry was also performed on tissue (Figure 1d). Among the cases successfully tested for microarray sections using corresponding antibodies: MYC rearrangement status (n = 344), 27 (16.3%) of p53 (DO-7; Dako, Carpinteria, CA, USA), MDM2 (IF2; 166 GCB-DLBCL cases and 13 (7.3%) of 177 ABC- Calbiochem, Billerica, MA, USA), Bcl-2 (Clone-124; DLBCL cases had MYC rearrangement/translocation. Dako), Ki-67 (MIB-1; Dako), pAKT (726E11; CST), Most but not all MYC-rearranged cases (MYC-R+) had Bcl-6 (PG-B6p; DAKO), FOXP1 (EPR4113; Abcam), high MYC-mRNA and Myc expression, with an MUM1/IRF4 (Dako), CD10 (56C6; Vantana), CD30 average percentage of Mychigh nuclei in the tumor (clone BerH2; Dako), BLIMP-1 (EPR16655; Epi- samples of 70.5% (Supplementary Figure S1A). We tomics), NF-κB subunits (Dako), CXCR4 (Abcam, thus set the cutoff for Myc overexpression (Mychigh) Cambridge, MA, USA), and survivin (EP2880Y; at ≥ 70%, so that Myc expression levels in MYC- Epitomics). BCL6 and BCL2 translocations and − non-rearranged (MYC-R )Mychigh patients compar- TP53 mutations were detected as described – ably ‘matched’ those in MYC-R+/Mychigh cases previously.15,21,24,35 42 (Supplementary Figure S1C) in comparisons of clinical and biological significance of Myc over- Gene Expression Profiling expression vs MYC translocation. Using this cutoff, we found that 175 (32.7%) of 535 Gene expression profiling for 457 patients was DLBCL patients, including 76 (27.9%) of 272 GCB- performed using the Affymetrix GeneChip Human DLBCL patients and 98 (37.8%) of 259 ABC-DLBCL Genome HG-U133 Plus 2.0 Array as described patients, were Mychigh (Figures 2a and b). Nineteen previously.21,24,35–37 The CEL files have been depos- (73%) of the 26 MYC-R+ GCB patients (one MYC-R+ ited in the National Center for Biotechnology GCB case had no expression data available) and 7 Information Gene Expression Omnibus repository (54%) of the 13 MYC-R+ ABC patients were Mychigh, (GSE31312). Microarray data were normalized for who had significantly higher MYC transcripts and further supervised clustering analysis. Multiple significantly worse survival compared with the t-tests were used to identify differentially expressed MYC-R+/Myclow cases (Figures 1e–g and Supple- genes and the P-values obtained were corrected for mentary Figures S1D). Compared with MYC-R− the false discovery rate using the β-uniform mixture patients, MYC-R+/Mychigh patients had significantly method. worse survival in GCB-DLBCL (P = 0.0001 for OS and

Modern Pathology (2015) 28, 1555–1573 MYC-associated biology and prognosis in DLBCL 1558 ZY Xu-Monette et al

in Diffuse Large B-cell Lymphoma in Germinal-center-B-cell-like in Activated-B-cell-like Diffuse Large B-cell Lymphoma Diffuse Large B-cell Lymphoma

Myc immunohistochemistry Myc immunohistochemistry Myc immunohistochemistry scores (%) scores (%) scores (%)

In Diffuse Large B-cell Lymphoma

Germinal-center Activated B-cell-like B-cell-like Months N=272 N=259 MYC-R+ GCB MYC-R+ ABC

Figure 1 (a–c) Histograms of Myc expression according to immunohistochemistry staining in all diffuse large B-cell lymphoma (DLBCL) patients, germinal center B-cell-like (GCB) DLBCL patients, and activated B-cell-like (ABC) DLBCL patients. (d) The mean Myc protein levels of ABC-DLBCL were significantly higher compared with those of GCB-DLBCL. (e and f) MYC rearrangement-positive (MYC-R+) DLBCL without high Myc immunohistochemistry scores (either GCB or ABC subtype) had significantly lower MYC-mRNA levels. (g) MYC-R+ DLBCL patients without high Myc immunohistochemistry scores had significantly better overall survival compared with MYC-R+/Mychigh DLBCL. Mychigh, high Myc protein expression; Myclow, low Myc protein expression.

Po0.0001 for PFS) but not in ABC-DLBCL (P = 0.56 For example, in GCB-DLBCL, Mychigh patients for OS and P = 0.14 for PFS) (Supplementary compared with Myclow patients more often had stage Figures S1E and F). Comparison of MYC expression III/IV disease, ≥ 2 extranodal sites, ECOG performance − levels between the MYC-R+ GCB, MYC-R GCB, status ≥ 2, tumor size ≥ 5 cm, International Prognostic − MYC-R+ ABC, and MYC-R ABC Mychigh groups Index 42, bone marrow involvement at clinical revealed that MYC-R+/Mychigh GCB-DLBCL had presentation, and less likely to have complete significantly higher Myc protein expression levels response. In ABC-DLBCL, Mychigh patients compared compared with all other subsets, and that among with Myclow patients had a higher proportion of + high both the GCB and ABC subtypes, MYC-R /Myc women, and did not show significant association with cases had a significantly higher level of MYC-mRNA − high other clinical parameters. The GCB and ABC subtypes levels compared with MYC-R /Myc cases of Mychigh patients differed significantly only in their (Figures 2c and d). frequencies of patients with age ≥ 60 years (Table 1). Similarly, in GCB-DLBCL, MYC-R+ patients were enriched in patients with adverse clinical features, Clinical Parameters Associated with Myc whereas in ABC-DLBCL, MYC rearrangement MYC Overexpression and Rearrangement was only associated with primary extranodal origin. The GCB subtype compared with the ABC subtype We analyzed the clinical parameters associated with + Myc overexpression (Table 1) and MYC rearrange- of MYC-R patients showed trends toward having ment (Supplementary Table 1). We further compared a higher proportion of patients with bone marrow MYC-R+/Mychigh and MYC-R−/Mychigh cases involvement and non-complete treatment response (Supplementary Table S2). Results of significant (Supplementary Table S1). characteristics are summarized in Supplementary In both the GCB- and ABC-DLBCL groups, MYC-R+/Mychigh patients appeared to have similar Table S3. Largely, both Myc overexpression and − MYC rearrangement could identify a subgroup of clinical features with MYC-R /Mychigh patients patients with adverse clinical features in GCB- (except complete response rate), yet may have DLBCL, whereas patients with these abnormalities elevated serum lactate dehydrogenase levels did not show such distinct characteristics among and higher frequency of extranodal origin ABC-DLBCLs. (Supplementary Table S2).

Modern Pathology (2015) 28, 1555–1573 MYC-associated biology and prognosis in DLBCL ZY Xu-Monette et al 1559

MYC-R+ Mychigh (27.9%) MYC-R+ Mychigh (37.8%) (16.3%) Myclow (72%) (7.3%) Myclow (62.2%)

GCB-DLBCL ABC-DLBCL

P=0.82

P=0.50

P=0.0075 P<0.0001

Mychigh GCB Mychigh ABC Mychigh GCB Mychigh ABC

Figure 2 Occurrence of MYC rearrangement (MYC-R) and overexpression (Mychigh) and MYC expression levels in diffuse large B-cell lymphoma (DLBCL). (a and b) Schematic diagrams showing the frequencies of MYC translocation (MYC-R+) and Myc overexpression (Mychigh) and their overlaps in germinal center B-cell-like (GCB) and activated B-cell-like (ABC) DLBCL in the current study cohort. (c and d) Comparisons of Myc protein and MYC-mRNA expression levels in DLBCL patients with or without MYC dysregulation. Among Mychigh groups, MYC-R+ GCB-DLBCL had highest Myc protein levels; MYC-R+ DLBCL (both GCB and ABC subtypes) had significantly higher MYC-mRNA levels compared with MYC-R− DLBCL. Note: Each dot represents one patient in the study cohort. MYC-R+, MYC rearrangement-positive; MYC-R−, MYC rearrangement-negative; Mychigh, high Myc protein expression/Myc overexpressing; Myclow, low Myc protein expression; MYC-RN/A, MYC rearrangement status not available.

Molecular Biomarkers Associated with Myc higher in the GCB subtype), and expression levels of Overexpression and MYC Rearrangement Bcl-2, p50, c-Rel (all higher in the ABC subtype), high low pAKT (higher in the GCB subtype), and cell-of- Myc DLBCL compared with Myc DLBCL, in origin-related biomarkers (Bcl-6, BLIMP-1, GCET-1, addition to having higher Myc levels and signifi- CD10, FOXP1, MUM1) (Table 1, Figure 3, and cantly higher frequencies of MYC translocation and Supplementary Table S3). ABC subtype (Table 1), had higher expression levels high Some of the pathobiological associations with of p53, MDM2, Bcl-2 (Myc ABC-DLBCL only), Mychigh DLBCL were shared by MYC-R+ GCB- Bcl-6, FOXP1, IRF4/MUM1 (Mychigh ABC-DLBCL − high DLBCL patients (compared with MYC-R GCB- only), Ki-67, pAKT, CXCR4, and CD10 (Myc DLBCL; Supplementary Tables S1 and S3). On the GCB-DLBCL only), but lower expression levels of other hand, in addition to the difference in Myc BLIMP-1 (in ABC-DLBCL only) and nuclear expres- activation mechanisms (MYC translocation or not), κ − sion of the NF- B subunits c-Rel and RelB (in ABC- MYC-R+/Mychigh compared with MYC-R /Mychigh DLBCL only, P = 0.019, figure not shown) (Figure 3). patients had higher frequencies of GCB subtype At the mRNA level, only MDM2 (in GCB-DLBCL and BCL2 translocation, and lower frequencies of only), FOXP1 (in ABC-DLBCL only), IRF4 (in ABC- BCL6 translocation, MUM1, CD30, and p52 expres- DLBCL only), and MME/CD10 transcript levels sion (Supplementary Table S2 and S3). Among GCB- showed corresponding correlations consistent with DLBCL patients, MYC-R+/Mychigh compared with those at the protein level (Figures 4a–d). PRDM1 MYC-R−/Mychigh patients had significantly higher mRNA levels were significantly lower in Mychigh expression levels of MDM2, CD10, and FOXP1 but a GCB-DLBCL compared with that in Myclow GCB- lower expression level of CD30; among ABC-DLBCL DLBCL (P = 0.0005). The GCB and ABC subtypes of patients, MYC-R+/Mychigh compared with MYC-R−/ Mychigh patients differed in frequencies of MYC Mychigh was associated with lower expression levels translocation, BCL2 translocation, TP53 mutation (all of Bcl-6 and pAKT (Figures 4f–l).

Modern Pathology (2015) 28, 1555–1573 MYC-associated biology and prognosis in DLBCL 1560 ZY Xu-Monette et al

Table 1 Clinicopathologic features of DLBCLa

DLBCL GCB-DLBCL ABC-DLBCL

P1 values P2 values P3 values P4 values Mychigh Myclow Mychigh Myclow Mychigh Myclow N (%) N (%) N (%) N (%) N (%) N (%)

Variables 175 (100) 360 (100) 76 (100) 196 (100) 98 (100) 161 (100) 0.016

Age (years) o60 73 (41.7) 150 (41.7) 1.0 42 (55.3) 91 (46.4) 0.22 30 (30.6) 57 (35.4) 0.50 0.0012 ≥ 60 102 (58.3) 210 (58.3) 34 (44.7) 105 (53.6) 68 (69.4) 104 (64.6)

Gender Female 78 (44.6) 146 (40.6) 0.38 30 (39.5) 85 (43.4) 0.56 48 (49.0) 59 (36.6) 0.05 0.21 Male 97 (55.4) 214 (59.4) 46 (60.5) 111 (56.6) 50 (51.0) 102 (63.4)

Stage I and II 64 (37.9) 179 (51.3) 0.0041 31 (41.9) 110 (58.5) 0.015 32 (34.0) 68 (43.0) 0.16 0.30 III and IV 105 (62.1) 170 (48.7) 43 (58.1) 78 (41.5) 62 (66.0) 90 (57.0)

B-symptoms No 103 (62.4) 227 (66.2) 0.41 45 (63.4) 133 (71.9) 0.19 58 (62.4) 91 (58.7) 0.57 0.89 Yes 62 (37.6) 116 (33.8) 26 (36.6) 52 (28.1) 35 (37.6) 64 (41.3)

LDH level Normal 58 (35.4) 137 (41.6) 0.18 24 (34.3) 82 (46.1) 0.091 34 (36.6) 54 (36.5) 0.099 0.76 Elevated 106 (64.6) 192 (58.4) 46 (65.7) 96 (53.9) 59 (63.4) 94 (63.5)

Number of extranodal sites 0–1 117 (69.6) 279 (80.9) 0.0045 45 (63.4) 156 (83.9) 0.0004 67 (69.8) 120 (76.9) 0.21 0.38 ≥ 2 51 (30.4) 66 (19.1) 26 (36.6) 30 (16.1) 29 (30.2) 36 (23.1)

ECOG performance status score 0–1 126 (77.3) 275 (86.8) 0.0082 53 (76.8) 150 (89.3) 0.013 72 (77.4) 122 (83.6) 0.24 0.93 ≥ 2 37 (22.7) 42 (13.2) 16 (23.2) 18 (10.7) 21 (22.6) 24 (16.4)

Size of largest tumor (cm) o5 64 (48.5) 170 (63.7) 0.0038 26 (47.3) 94 (64.4) 0.028 38 (49.4) 74 (62.2) 0.076 0.81 ≥ 5 68 (51.5) 97 (36.3) 29 (52.7) 52 (35.6) 39 (50.6) 45 (37.8)

IPI score 0–2 88 (51.5) 234 (67.4) 0.0004 39 (52.7) 139 (74.3) 0.0007 47 (49.0) 92 (58.6) 0.13 0.63 3–5 83 (48.5) 113 (32.6) 35 (47.3) 48 (25.7) 49 (51.0) 65 (41.4)

Therapy response CR 117 (66.9) 285 (79.2) 0.002 46 (60.5) 155 (79.1) 0.0018 70 (71.4) 127 (78.9) 0.17 0.13 PR 33 38 24 22 21 16 SD 9 15 5 10 4 5 PD 16 22 1 9 3 13

Primary origin LN DLBCL 111 (63.8) 227 (63.9) 0.97 47 (63.5) 125 (64.8) 0.85 61 (63.5) 100 (62.9) 0.92 1.00 EN DLBCL 63 (36.2) 128 (36.1) 27 (36.5) 68 (35.2) 35 (36.5) 59 (37.1)

Bone marrow involvement No 130 (83.9) 287 (92.6) 0.0036 52 (81.3) 163 (95.3) 0.0006 77 (85.6) 122 (89.1) 0.43 0.48 Yes 25 (16.1) 23 (7.4) 12 (18.8) 8 (4.7) 13 (14.4) 15 (10.9)

Ki-67 index (%) o70 40 (22.9) 149 (42.2) o0.0001 24 (31.6) 83 (43.2) 0.097 16 (16.3) 65 (40.6) o0.0001 0.028 ≥ 70 135 (77.1) 204 (57.8) 52 (68.4) 109 (56.8) 82 (83.7) 95 (59.4)

TP53 mutations No 114 (74.5) 252 (78.8) 0.35 43 (65.2) 131 (75.7) 0.10 71 (81.6) 118 (81.9) 0.95 0.02 Yes 39 (25.5) 68 (21.3) 23 (34.8) 42 (24.3) 16 (18.4) 26 (18.1)

MYC translocation No 81 (75.7) 220 (94.4) o0.0001 23 (54.8) 113 (94.2) o0.0001 58 (89.2) 106 (94.6) 0.23 o0.0001 Yes 26 (24.3) 13 (5.6) 19 (45.2) 7 (5.8) 7 (10.8) 6 (5.4)

BCL2 translocation No 121 (86.4) 230 (79.9) 0.11 38 (67.9) 106 (67.9) 1.00 83 (98.8) 123 (93.9) 0.09 o0.0001 Yes 19 (13.6) 58 (20.1) 18 (32.1) 50 (32.1) 1 (1.2) 8 (6.1)

BCL6 translocation No 77 (64.2) 169 (68.7) 0.38 37 (71.2) 103 (75.7) 0.52 39 (58.2) 65 (59.6) 0.85 0.14 Yes 43 (35.8) 77 (31.3) 15 (28.8) 33 (24.3) 28 (41.8) 44 (40.4)

Bcl-2 expression o70% 76 (43.7) 195 (55.7) 0.012 40 (53.3) 121 (63.4) 0.16 35 (35.7) 73 (46.2) 0.12 0.03 ≥ 70% 98 (56.3) 155 (44.3) 35 (46.7) 70 (36.6) 63 (64.3) 85 (53.8)

Abbreviations: ABC, activated-B-cell-like; CR, complete response; DLBCL, diffuse large B-cell lymphoma; ECOG, Eastern Cooperative Oncology Group; EN, extranodal; GCB, germinal-center-B-cell-like; IPI, International Prognostic Index; LDH, lactate dehydrogenase; LN, lymph node; Mychigh, high Myc protein expression; Myclow, low Myc protein expression; PD, progressive disease; PR, partial response; SD, stable disease. Note: P-values indicate the significance of differences in the positivity frequencies of the listed parameters between two groups. P1 values are for high low high low comparisons between overall Myc and Myc DLBCL patients; P2 values are for comparisons between Myc and Myc GCB-DLBCL high low high patients; P3 values are for comparisons between Myc and Myc ABC-DLBCL patients; and P4 values are for comparisons between Myc GCB-DLBCL and Mychigh ABC-DLBCL patients. For therapy response, we calculated P-values for differences between CR and other responses. aWith high or low Myc expression levels (Mychigh vs Myclow) in the overall, GCB, and ABC cohorts. Bold values are statistically significant.

Modern Pathology (2015) 28, 1555–1573 MYC-associated biology and prognosis in DLBCL ZY Xu-Monette et al 1561

Prognostic Effect of Myc Overexpression with or and Supplementary Table S4B) only showed a few without MYC Translocations and Associated Gene differentially expressed genes below false discovery Expression Profiling Signatures rate threshold of 0.30 (not including MYC), most of which are involved in cell proliferation, gene We combined the survival analysis with gene expression, ribosome biogenesis, and metabolism, expression profiling comparisons to assess the role suggesting the presence of heterogeneity and post- of MYC abnormalities in DLBCL and to study 13,22–24 transcriptional regulation of MYC as a cause of Myc potential mechanisms. As in earlier studies, overexpression within the MYC-R−/Mychigh GCB- in the present study, high expression levels of Myc DLBCL group. The comparison between MYC-R+ was associated with significantly worse OS and PFS GCB-DLBCL and MYC-R− GCB-DLBCL overall is in both GCB- and ABC-DLBCL (Supplementary shown in Supplementary Figure S4E. Figure S2). Similar prognostic impact was also In contrast, in ABC-DLBCL, the prognostic signi- shown by high MYC-mRNA levels in GCB- and ficance (Figure 5d) and gene expression profiling ABC-DLBCL (Supplementary Figures S3A–D). Gene high features of Myc overexpression did not depend on expression profiling analysis showed that Myc MYC translocation. MYC-R−/Mychigh ABC-DLBCL GCB- and ABC-DLBCL had different gene expression had significantly poorer survival compared with profiling signatures (Table 2, Supplementary Figures the overall Myclow or MYC-R−/Myclow ABC-DLBCL S2D and F, and Supplementary Table S4A). The (Figure 5d and Supplementary Figures S4G–I) and significantly differentially expressed genes between distinct gene expression profiling signatures (false these two groups included CDCA7L, IGF2BP3, and discovery rate o0.01; Figure 5e and Supplementary RUVBL2, which are known to have roles in the Table S4C). The genes in the MYC-R−/Mychigh ABC- oncogenic transformation by MYC or to interact DLBCL gene expression profiling signature, over- with MYC. lapped with differentially expressed genes between MYC rearrangement did not show prognostic the overall Mychigh vs Myclow ABC-DLBCL patients, significance in the Myclow cases [for OS: P = 0.25 + low included typical Myc targets mainly related to (OS of MYC-R /Myc patients was slightly better cell proliferation, the cell cycle, gene expression, − low than MYC-R /Myc DLBCL); for PFS: P = 0.71]. ribosome biogenesis, metabolism (Table 4), and high Dividing the Myc cases with unfavorable prog- cooperating oncogenes such as RUVBL2, as well as + high − high nosis into MYC-R /Myc and MYC-R /Myc two IGF2BP3 involved in post-transcriptional regulation types and comparing their prognosis and gene of MYC, and HINT1 modulating p53 levels and expression profiling features, we further found that the p53 pathway. Different from the MYC-R−/Mychigh + high − high MYC-R /Myc GCB-DLBCL and MYC-R /Myc ABC-DLBCL cases, MYC-R+/Mychigh ABC-DLBCL ABC-DLBCL were the main contributors to the compared with overall Myclow ABC-DLBCL only overall worse prognosis and distinct gene expression showed nonsignificant trends towards worse OS and high profiling signatures of the Myc GCB-DLBCL and PFS (Figure 5d and Supplementary Figures S4G–I) high Myc ABC-DLBCL groups, respectively. and only a few differentially expressed genes (false + high In the GCB-DLBCL group, MYC-R /Myc discovery rate o0.30, Supplementary Figure S4F). patients had significantly worse survival than both − No genes were found differentially expressed Myclow and MYC-R /Mychigh patients did. Although + high − between MYC-R /Myc ABC-DLBCL and MYC-R MYC-R /Mychigh GCB-DLBCL showed trends −/Mychigh ABC-DLBCL, or between MYC-R+/Mychigh towards worse survival compared with the overall ABC-DLBCL and the overall MYC-R− ABC-DLBCL Myclow GCB-DLBCL (P = 0.40 for OS, Figure 5a; group. This is in contrast with the distinct gene P = 0.48 for PFS; Supplementary Figure S4A), and + high − expression profiling feature of MYC-R /Myc the MYC-R /Myclow GCB-DLBCL (for OS: P = 0.32; GCB-DLBCL shown in Figure 5b and Supple- Supplementary Figure S4C; for PFS: P = 0.21), mentary Figures S4E. Comparison between overall the differences were not significant. Biologically, + high + high − MYC-R /Myc ABC-DLBCL and MYC-R /Myc only MYC-R+/Mychigh (but not MYC-R /Mychigh) GCB-DLBCL indicated their different and potentially GCB-DLBCL compared with Myclow GCB-DLBCL heterogeneous tumor biology (Figure 5f) (between showed a distinct gene expression profiling signa- MYC-R+/Mychigh and MYC-R+/Myclow GCB-DLBCL, ture (false discovery rate o0.01) (Figure 5b, or between MYC-R+/Mychigh and MYC-R+/Myclow Supplementary Table S4B, and Table 3), and this ABC-DLBCL, we did not find significant dif- signature mostly overlapped the Mychigh gene ferentially expressed genes below false dis- expression profiling signature identified in the over- covery rate thresholds of 0.05–0.50). MYC-R+ ABC- all GCB-DLBCL group (Table 2 and Supplementary DLBCL appears to have decreased B-cell receptor Figure S2D) involving cell proliferation, gene expres- signaling compared with MYC-R+ GCB-DLBCL (false sion, metabolism, apoptosis, microenvironment and discovery rate o0.30; Table 4 and Supplementary immune response, and microRNA genes. In contrast, Table S4C). However, the ‘loss’ of the gene expres- − the MYC-R /Mychigh GCB-DLBCL patients compared sion profiling signature may also be due to the small with Myclow GCB-DLBCL (Supplementary Figures case number. S4D, Table 3, and Supplementary Table S4B) or Comparing between GCB and ABC subtypes of MYC-R+/Mychigh GCB-DLBCL (Figure 5c, Table 3, Mychigh patients, the overall GCB and ABC subtypes

Modern Pathology (2015) 28, 1555–1573 MYC-associated biology and prognosis in DLBCL 1562 ZY Xu-Monette et al

P P=0.63 =0.12 P=0.0002 P =0.24

Myc Myc Myc Myc Myc Myc Myc Myc Myc Myc Myc Myc Myc Myc Myc Myc

GCB ABC GCB ABC GCB ABC GCB ABC

P<0.0001 P=0.0002 P<0.0001 P<0.0001

Myc Myc Myc Myc Myc Myc Myc Myc Myc Myc Myc Myc Myc Myc Myc Myc

GCB ABC GCB ABC GCB ABC GCB ABC

P=0.012 P=0.01 P=0.013 P =0.85

Myc Myc Myc Myc Myc Myc Myc Myc Myc Myc Myc Myc Myc Myc Myc Myc

GCB ABC GCB ABC GCB ABC GCB ABC

Figure 3 Expression of pathobiological markers in germinal center B-cell-like (GCB) and activated B-cell-like (ABC) diffuse large B-cell lymphoma with or without Myc overexpression. In both GCB- and ABC-DLBCL, Myc overexpression was associated with significantly higher levels of p53 (a), MDM2 (b), Ki-67 (d), Bcl-6 (f), and FOXP1 (g), and a significantly lower level of c-Rel (k). In GCB-DLBCL only, Myc overexpression was associated with significantly higher levels of CD10 (e), pAKT (j), and CXCR4 (l). In ABC-DLBCL only, Myc overexpression was associated with significantly higher levels of Bcl-2 (c) and MUM1 (h) levels and a significantly lower level of BLIMP-1 (i). Mychigh, high Myc protein expression; Myclow, low Myc protein expression.

of Mychigh patients showed no significant difference lymphomagenesis may have a role in defining its in survival (ABC subtype showed nonsignificant biological feature. trends towards poorer survival; Supplementary Figures S3E and F). However, MYC-R+/Mychigh GCB-DLBCL showed unfavorable trends compared high − Concurrent Evaluation of Myc/Bcl-2 Overexpression with Myc ABC-DLBCL (either MYC-R or and MYC Translocations MYC-R+). The P-valueforthedifferenceinPFS between the MYC-R+/Mychigh GCB-DLBCL and We examined whether the prognostic value of Myc MYC-R−/Mychigh ABC-DLBCL patients was 0.058 overexpression is contributed by or depends on the (Figure 5g). MYC-R+/Mychigh GCB-DLBCL compared molecular marker associations with Mychigh DLBCL with MYC-R−/Mychigh ABC-DLBCL or Myclow ABC- as shown in Figure 3. As shown previously, the DLBCL showed distinct gene expression profiling prognostic significance of Mychigh and Bcl-2high in – signatures overlapping with the one comparing with DLBCL significantly depend on each other.13,20,23 26 MYC-R− GCB-DLBCL. Comparisons between MYC-R+ In addition, to a certain extent, the prognostic DLBCL and MYC-R− DLBCL overall (regardless of significance of Mychigh showed dependence on Mychigh or Myclow, GCB or ABC), and between MYC-R+/ CXCR4,40 FOXP1, and MUM1 overexpression, Mychigh DLBCL overall and MYC-R−/Mychigh DLBCL which are also associated with Bcl-2 overexpression overall (regardless of GCB or ABC) are shown in (the association of MUM1 was only in ABC but Figures 5h and i (Table 5 and Supplementary Table not in GCB), high Ki-67 (for OS but not for PFS), S4D). These analyses suggest the distinctive biology of and low BLIMP-1 expression (Supplementary MYC-R+/Mychigh GCB-DLBCL and cell of origin during Figure S5).

Modern Pathology (2015) 28, 1555–1573 MYC-associated biology and prognosis in DLBCL ZY Xu-Monette et al 1563

Myc Myc Myc Myc Myc Myc Myc Myc Myc Myc Myc Myc Myc Myc Myc Myc n=151 n=57 n=129 n=84 n=151 n=57 n=129 n=84 n=151 n=57 n=129 n=84 n=151 n=57 n=129 n=84

GCB ABC GCB ABC GCB ABC GCB ABC

Figure 4 (a–d) Myc overexpression was correlated with significantly higher levels of MDM2, IRF4, and MME/CD10 mRNA expression in germinal center B-cell-like (GCB) diffuse large B-cell lymphoma and significantly higher levels of FOXP1 and IRF4 mRNA expression in activated B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL). (e) The MYC-R+/Mychigh compared with MYC-R−/Mychigh group had significantly lower levels of MUM1 expression. (f–j) The MDM2, CD30, CD10, and FOXP1 levels in Mychigh GCB-DLBCL patients with MYC translocation were significantly different from those of Mychigh GCB-DLBCL patients without MYC translocation. (k and l) The Bcl-6 and pAKT levels of Mychigh ABC-DLBCL patients with MYC translocation were significantly lower compared with those of Mychigh ABC- DLBCL patients without MYC translocation. Mychigh, high Myc protein expression; Myclow, low Myc protein expression; MYC-R+, MYC rearrangement-positive; MYC-R−, MYC rearrangement-negative; MYC-RN/A, MYC rearrangement status not available.

Table 2 Gene expression profile signatures of Myc protein overexpression in DLBCL, GCB-DLBCL, and ABC-DLBCL

DLBCL GCB-DLBCL ABC-DLBCL

Mychigh vs Myclow (false discovery Mychigh vs Myclow (false discovery rate Mychigh vs Myclow (false discovery rate o0.05, fold change 41.53) o0.01, fold change 41.68) rate o0.01, fold change 41.31)

Upregulated MYC, AICDA, SNHG1, SNHG4, MYC, PEG10, SNHG4, STRBP, CYP39A1, MYC, MAD2L1, EEF1E1, RUVBL2, TMEM97, PAICS, TCL1A, XK, DKFZp686O24166, PAICS, FAM72A/B/ SNHG1, RG9MTD1, MRPL3, IPO7, FAM129C, CKS2, PEG10, C/D, CDC25A, SLC16A1, RPS21, DEPDC1, CCDC86, TFAM, GAR1, MATR3, IGF2BP3, SLC16A1, FAM72A/ HSPD1 SNHG4, TOMM5, NOC3L, WDR43, B/C/D, HELLS, CDCA7L, DDX21, LYAR, RPL24, LOC388796, MAD2L1, MRPL3, PRO2964, SNHG8, WDR75, DCTPP1, MAT2A, C13orf18, MIR17HG QDPR, APEX1

Downregulated CD3E, HOPX, TRBC1, COL3A1, MIR155HG, TRBC1, GABBR1/UBD, CD58, RGS1, COL3A1, TRBC1, ITM2A, CD3E, BHLHE41, GZMK, DUSP4, TRBC1, GZMK, ITGB5, ITM2A SLAMF7, LCP2, RGS1, ITM2A, SKI, SLAMF8, CD44, LOC285628, FYB, CCL5, MDFIC, CCND2, BCL11B, TNFAIP3, SLFN5, SNX9, IL10RA, GBP2

Abbreviations: ABC, activated B-cell-like; DLBCL, diffuse large B-cell lymphoma; GCB, germinal center B-cell-like; Mychigh, high Myc protein expression; Myclow, low Myc protein expression. Note: Genes are listed by the order of fold change (high to low).

Modern Pathology (2015) 28, 1555–1573 MYC-associated biology and prognosis in DLBCL 1564 ZY Xu-Monette et al

Germinal-center-B-cell-like

Activated-B-cell-like

Months

Figure 5 Combined prognostic and biologic analysis of germinal-center-B-cell-like (GCB) and activated-B-cell-like (ABC) diffuse large B-cell lymphoma (DLBCL) with or without MYC translocation (MYC-R) and/or Myc overexpression (Mychigh) in the current study cohort. (a) GCB-DLBCL patients with both MYC translocation and Myc overexpression (MYC-R+/Mychigh) had significantly worse overall survival compared with GCB-DLBCL patients with low Myc expression (Myclow) and Mychigh patients without MYC translocation (MYC-R−/ Mychigh). The MYC-R−/Mychigh group did not have significant poorer survival compared with the Myclow group. (b) Genes significantly differentially expressed between MYC-R+/Mychigh GCB-DLBCL patients and Myclow GCB-DLBCL patients (false discovery rate o0.01, fold change 42.38). (c) Genes significantly differentially expressed between MYC-R+/Mychigh and MYC-R−/Mychigh GCB-DLBCL patients (false discovery rate o0.30). (d) Only MYC-R−/Mychigh but not MYC-R+/Mychigh ABC-DLBCL patients had significantly OS compared with Myclow ABC-DLBCL patients. (e) Genes significantly differentially expressed between MYC-R−/Mychigh ABC-DLBCL and Myclow ABC- DLBCL (false discovery rate o0.01, fold change 41.57). (f) Genes significantly differentially expressed between ABC and GCB subtypes of MYC-R+ DLBCL. (g) MYC-R+/Mychigh GCB-DLBCL showed trend towards worse progression-free survival compared with MYC-R−/Mychigh ABC-DLBCL patients with a borderline P-value. (h) Genes significantly differentially expressed between MYC-R+ and MYC-R−DLBCL (false discovery rate o0.01, fold change 41.66). (i) Genes significantly differentially expressed between MYC-R+/Mychigh and MYC-R−/Mychigh DLBCL (false discovery rate o0.01, fold change 42.05). ABC, activated-B-cell-like; GCB, germinal-center-B-cell-like; Mychigh, high Myc protein expression; Myclow, low Myc protein expression; MYC-R+: MYC rearrangement-positive; MYC-R−, MYC rearrangement-negative.

The lack of prognostic significance of Myc MYC-R+/Mychigh ABC-DLBCL, or MYC-R+/Myclow overexpression without MYC translocation in GCB- cases (Supplementary Figures S6C and D); however, DLBCL (Figure 5a) could be attributable to we could not distinguish whether the synergy in the favorable prognosis of Mychigh/Bcl-2low GCB- GCB is with Bcl-2 protein or with BCL2 transloca- DLBCL patients (Figures 6a and b and Supple- tion. Compared with Myclow, MYC-R−/Mychigh GCB- mentary Figures S6A and B). In contrast, in the DLBCL, or overall MYC-R− GCB-DLBCL, MYC-R+/ ABC-DLBCL group, most MYC-R−/Mychigh patients Mychigh GCB-DLBCL showed nonsignificant trends (64%) also had Bcl-2 overexpression, which con- toward higher Bcl-2 levels (P = 0.34, 0.27, and tributed to this group’s worse survival. Concurrent P = 0.17, respectively; figures not shown; Supple- Bcl-2 overexpression also had significant prognostic mentary Tables S1 and 2). The gene expression impact in MYC-R+/Mychigh GCB-DLBCL (Figures 6c profiling analysis revealed no significant differen- and d), but not in overall MYC-R+ ABC-DLBCL, tially expressed genes between MYC-R+ GCB-DLBCL

Modern Pathology (2015) 28, 1555–1573 MYC-associated biology and prognosis in DLBCL ZY Xu-Monette et al 1565

Table 3 Gene expression profile signatures of Myc protein overexpression in GCB-DLBCL

Functional categories Upregulated Downregulated

1. MYC-R+/Mychigh vs Myclow GCB-DLBCL (false discovery rate o0.01, fold change 42.38) Signaling TRAF1, DUSP4, GABBR1/ UBD Cell proliferation and growth, gene expression MYC, SMAD1, BACH2, STRBP FAM129A Metabolism NNMT Cell death PEG10, ZNF385B BCL2A1, TMEM49 Immune response, anti-viral/anti-microbial activities IGJ, DKFZp686O24166/ NCR3LG1 CD58, GBP1, SLAMF7, LYZ Cell adhesion, extracellular matrix, migration FN1, BGN, CD44 microRNAs MIR21, MIR155HG Unknown function TPD52

2. MYC-R−/ Mychigh vs Myclow GCB-DLBCL (false discovery rate o0.30, fold change 41.2) Signaling RGS8, GPS1, FAM123A, PDLIM7 Cell proliferation and growth, gene expression, C9orf100, SMARCA4, ZNF8, MRPS12, ribosome biogenesis EMG1, INTS1 Metabolism SLC25A27, FADS2, ACAD9 Microtubules, migration, cell interaction TUBB2C, TUBB3 LGALS8 Transport ABCA4, CHCHD4 VPS36 Long noncoding RNA, RNA gene NAPSB LOC202181, LOC440944

3. MYC-R+/Mychigh vs MYC-R−/Mychigh GCB-DLBCL (false discovery rate o0.30, fold change 41.3) Signaling SIKE1 SPRED1 Transcription, ribosome biogenesis NAF1, RRP1B, SMAD1 FOXN3, ATN1 Metabolism GANC Extracellular matrix, migration, cytoskeleton BGN, TRIOBP Unknown function PWWP2A KIAA0913

Abbreviations: DLBCL, diffuse large B-cell lymphoma; GCB, germinal-center-B-cell-like; MYC-R+, MYC rearrangement-positive; MYC-R−, MYC rearrangement-negative; Mychigh, high Myc protein expression; Myclow, low Myc protein expression.

Table 4 Gene expression profile signatures of Myc overexpression in ABC-DLBCL

Functional categories Upregulated Downregulated

1. MYC-R − / Mychigh vs Myclow ABC-DLBCL (false discovery rate o0.01, fold change 41.57) Cell proliferation, cell cycle, gene MYC, IGF2BP3, FOXP1, CCNB1, CKS2, DCAF13, THOC4, DDX11, expression, ribosome biogenesis RUVBL2, RPS15, RPLP0, RPL35, RPL27A, RPLP2, RPSA, RPS21, RPLP1, RPL15, PABPC1, DNAJC2, MAD2L1 Metabolism GRHPR, CYB5R2, ESD, TMEM97 DNA damage response EEF1E1, HINT1 Transport CSE1L, IPO5 RNA gene; unknown function SNHG1, LOC100291837

2. MYC-R+/ Mychigh vs Myclow ABC-DLBCL (false discovery rate o0.30, fold change 41.43) Cell cycle, gene expression, ribosome MYC, RPL24, NAF1, EIF4B, CCNT1, RPL29, ZNF485 biogenesis Metabolism GART, PLA2G12A, MDH1B Proteasome degradation, transport STUB1, NXT2 Pseudogene RPS10P5

3. MYC-R+ ABC-DLBCL vs MYC-R+ GCB-DLBCL (false discovery rate o0.15, fold change 41.3) Signaling TNFRSF13B, SERPINA1, SH3BP5, TNIP3, ENTPD1, P2RY10, STAP1, MME CNPY3, PGAP2 Transcription, ribosome biogenesis BATF, MNDA, RUNX1 MYBL1 Metabolism C6orf150, KIAA0467 Extracellular matrix, migration, IQGAP2, ARPC5, TMSB10, ACTA2, PARVB MARCKSL1 cytoskeleton Immune response LILRB1/2, CD47 CAMP Transport, degradation NXT2, EXOC4, PTPN1 Unknown function GRAMD1B, PHACTR2 C8orf6, TPD52

Abbreviations: ABC, activated-B-cell-like; DLBCL, diffuse large B-cell lymphoma; MYC-R+, MYC rearrangement-positive; MYC-R−, MYC rearrangement-negative; Mychigh, high Myc protein expression; Myclow, low Myc protein expression.

Modern Pathology (2015) 28, 1555–1573 MYC-associated biology and prognosis in DLBCL 1566 ZY Xu-Monette et al

Table 5 Gene expression profile signatures of MYC-R+ GCB-DLBCL and MYC-R+ DLBCL overall

Functional categories Upregulated Downregulated

1. All (Mychigh or Myclow) MYC-R+ GCB vs all (Mychigh or Myclow) MYC-R − GCB (false discovery rate o0.01, fold change 42.3) Signaling TRAF1, DUSP4 Cell proliferation and growth, gene expression MYC, SMAD1, STRBP, BACH2 LMO2, FAM129A, STAT3 Apoptosis ZNF385B, PEG10 FAS, BCL2A1, TMEM49 Immune response, anti-viral/ anti-microbial IGJ, DKFZp686O24166/NCR3LG1 CD58, LYZ, CHI3L1 activities Cell adhesion, extracellular matrix, migration BGN, CD44 MicroRNA, long noncoding RNA MIR155HG, MIR21, NCRNA00152/LINC00152 Unknown function LOC283027

2. All (GCB or ABC) MYC-R+ vs all (GCB or ABC) MYC-R− (false discovery rate o0.01, fold change 41.66) Signaling BMP3 TNFAIP3 Cell proliferation and growth, gene expression MYC, BACH2, STRBP, SMAD1 Apoptosis PEG10, ZNF385B BCL2A1, CFLAR, TMEM49 Metabolism CYP39A1, PLA2G12A Immune response DKFZp686O24166/NCR3LG1 Cell adhesion, extracellular matrix, migration PCDH9 CD44 MicroRNA, long noncoding RNA GAS5 MIR155HG, NCRNA00152/ LINC00152, MIR21 Transport SLC25A27, SLC44A1, SLC35E3 Degradation RFFL Unknown function C4orf34 LOC283027

3. MYC-R+/Mychigh DLBCL (GCB or ABC) vs MYC-R−/Mychigh DLBCL (GCB or ABC) (false discovery rate o0.01, fold change 42.05) Signaling BMP3, BMP7 Transcription, ribosome biogenesis MYC, IKZF2, SMAD1, STRBP STAT3, BATF Apoptosis TMEM49, CFLAR Immune response DKFZp686O24166/NCR3LG1 Cell adhesion, extracellular matrix, migration TAPT1, PCDH9 EMILIN2, TPM4, ARHGAP25, CD44 MicroRNA, long noncoding RNA MIR21, LINC00152/NCRNA00152 Transport SLC35E3 Unknown function LOC100288765, LOC283027

Abbreviations: ABC, activated B-cell-like; DLBCL, diffuse large B-cell lymphoma; GCB, germinal center B-cell-like; MYC-R+, MYC rearrangement- positive; MYC-R−, MYC rearrangement-negative; Mychigh, high Myc protein expression; Myclow, low Myc protein expression.

with Bcl-2 expression and MYC-R+ GCB-DLBCL survival of MYC-R+ double-positive lymphoma without Bcl-2 expression. Correspondingly, patients patients with the GCB or ABC subtypes appeared to with MYC/BCL2 double-hit lymphoma and those be different, although P-values were not significant with single MYC rearrangements did not have and the case numbers were small. significant differentially expressed genes, which is consistent with an earlier study.16 The survival of MYC-R+ Mychigh/Bcl-2high GCB-DLBCL patients was Discussion markedly poorer compared with that of MYC-R− Mychigh/Bcl-2high GCB-DLBCL patients but this Previously, we reported MYC translocation and difference was not significant (Figures 6e and f). Myc overexpression as adverse prognostic biomarkers However, this difference was significant when individually.15,24 In this study, we analyzed the we used cutoffs of ≥ 40% or 450% for Myc occurrence of MYC translocation and Myc over- overexpression. expression in GCB- and ABC-DLBCL (Figures 2a and Although concurrent evaluation of Myc and Bcl-2 b), and compared with the clinical features and tumor expression improves the specificity of Myc biomar- biology associated with these two overlapping bio- ker in DLBCL, MYC rearrangement continues to markers which have not been done by previous demonstrate clinical value. In our cohort, there was studies, and examined the dependence/independence no significant difference in survival between patients between their indicated prognoses. To reduce the with the GCB or ABC subtypes of MYC-R− Mychigh/ difference in Myc expression levels as a causing factor Bcl-2high (ie, double-positive lymphoma). Among for the differential prognostic effect and tumor biology overall double-positive lymphoma patients, patients between MYC translocation and Myc overexpression with MYC-R+ GCB double-positive lymphoma activated by other mechanisms, the cutoff for Mychigh patients had significantly poorer survival compared was set at ≥ 70% in this study, which is the optimal with those double-positive lymphoma patients with- cutoff for predicting MYC translocation according to out MYC rearrangement (Figures 6g and h). The previous studies.6,7,31 Using this cutoff, the frequency

Modern Pathology (2015) 28, 1555–1573 MYC-associated biology and prognosis in DLBCL ZY Xu-Monette et al 1567

MYC-R¯ / Mychigh MYC-R¯ / Myc high GCB-DLBCL GCB-DLBCL

MYC-R+ / Mychigh MYC-R+ / Mychigh GCB-DLBCL GCB-DLBCL

Mychigh Bcl-2high Mychigh Bcl-2high GCB-DLBCL GCB-DLBCL

Mychigh Bcl-2high Mychigh Bcl-2high

Months Months

Figure 6 Prognostic analysis in Mychigh germinal-center-B-cell-like (GCB) diffuse large B-cell lymphoma (DLBCL) and in MychighBcl-2high DLBCL patients. (a and b) Among MYC-non-rearranged (MYC-R−) Mychigh GCB-DLBCL patients, those with Bcl-2 overexpression had significantly worse overall survival and progression-free survival compared with those who did not have Bcl-2 overexpression. (c and d) Among patients with MYC-R+/Mychigh GCB-DLBCL, those with Bcl-2 overexpression had significantly poorer progression-free survival compared with those who did not have Bcl-2 overexpression. The P-value for overall survival was not significant. (e and f) Among patients with MychighBcl-2high GCB-DLBCL, those with MYC rearrangement had poorer overall and progression-free survival compared with those without MYC rearrangement did, but these differences were not statistically significant. (g and h) Among MychighBcl-2high DLBCL patients, GCB-DLBCL patients with MYC rearrangement had significantly poorer overall and progression-free survival than MychighBcl-2high patients without MYC rearrangement did. ABC, activated-B-cell-like; Bcl-2high, high Bcl-2 protein expression; Bcl-2low, low Bcl-2 protein expression; GCB, germinal-center-B-cell-like; Mychigh, high Myc protein expression; MYC-R+, MYC rearrangement-positive; MYC-R−, MYC rearrangement-negative. of Myc overexpression was 32.7% in overall DLBCL MYC translocation and Myc overexpression in DLBCL patients (close to the frequencies by other indepen- only partially overlaps and evaluation of both is critical dent studies13,22,23), 73% in MYC-R+ GCB-DLBCL, for stratifying patients and predicting treatment and 54% in MYC-R+ ABC-DLBCL (lower than the outcomes. MYC-rearranged DLBCL without Myc 93% in Green et al6 and 85% in Horn et al10). protein overexpression did not show significantly Tables 6–7 summarize the results of published MYC worse survival (Figure 1g). However, evaluation of studies including ours. The current study shows that Myc overexpression alone is also insufficient for

Modern Pathology (2015) 28, 1555–1573 MYC-associated biology and prognosis in DLBCL 1568 ZY Xu-Monette et al

predicting poorer prognosis in MYC-R− cases, espe- translocations),51,52 immunoregulation,53 and ionizing cially in MYC-R− GCB-DLBCL (Figure 5a). This low radiation-induced double-strand break signaling.54 In specificity for Mychigh as an adverse prognostic addition, in MYC-R+/Mychigh GCB-DLBCL, antiapop- factor can be improved by concurrent evaluation of totic PEG10 and ZNF385B, which modulates p53 Bcl-2 expression (Supplementary Figures S5 and S6), activity resulting in cell-cycle arrest over apoptosis, which is overexpressed mainly in ABC-DLBCL and were significantly upregulated. associated with Myc overexpression. However, the One possible reason for the differential prognoses survival of MYC-rearranged MychighBcl-2high GCB and gene expression profiling signatures among GCB double-positive lymphoma patients remains signifi- or ABC subtype of Mychigh and MYC-R+ cases is the cantly worse than other double-positive lymphoma difference in Myc protein levels. For example, MYC- patients (Figures 6g and h). R+/Mychigh GCB-DLBCL had highest Myc expression The biological investigation (in this regard, ≥ 70% levels (Figure 2c) and significant or nonsignificant is a better cutoff compared with ≥ 40% for Mychigh in trends towards worse prognosis compared with all our cohort) revealed that MYC activation was other three Mychigh groups. MYC-R+ cases with low associated with significantly increased or decreased Myc protein expression had good prognosis even expression of genes and proteins involved in cell though having an MYC rearrangement. Different proliferation (e.g., pAKT, Ki-67), apoptosis (p53, MYC translocation partners, and/or breakpoints out- Bcl-2, FAS, BCL2A1, PEG10, HINT1, TRAF1), differen- side of MYC gene leaving MYC repressor element tiation (PRDM1, BLIMP-1, BACH2 (which represses intact during rearrangement,4,17 may cause the low PRDM1)43), noncoding RNAs (eg, LINC00152, MYC-mRNAs in these MYC-R+/Myclow DLBCL cases GAS5, SNHG1, NAPSB) and microRNAs, microenvi- (Figures 1e–g). Compared with MYC-R+/Mychigh ronment, and immune responses, as well as cell- GCB-DLBCL, ABC subtype of MYC-R+/Mychigh of-origin markers (Figure 3 and Tables 3–5). DLBCL had similar MYC-mRNA but significantly Corresponding to the differences in prognostic effect lower Myc protein levels (Figure 2d) and trends of between various Mychigh subtypes, only MYC-R+/ better survival. MYC-R−/Mychigh GCB-DLBCL com- Mychigh GCB-DLBCL and MYC-R−/Mychigh ABC- pared with Myclow GCB-DLBCL had significantly DLBCL, but not MYC-R−/Mychigh GCB-DLBCL or higher levels of MYC-mRNA and Myc protein, but MYC-R+/Mychigh ABC-DLBCL, demonstrated distinct the false discovery rate for MYC upregulation in gene gene expression profiling feature compared with expression profiling analysis was high (40.45), the Myclow subgroup (Figures 5b and e and Supple- which may suggest either Mychigh or Myclow group mentary Figures S4D and F). Remarkably, MYC-R+/ are heterogeneous and the molecular mechanisms Mychigh GCB-DLBCL had a characteristic gene expres- inducing Myc in this Mychigh group include sion profiling in DLBCL. Myc activation was asso- post-transcriptional regulation. A previous Myc ciated with gene expression profiling signatures study in immature and mature GCB cells during suggesting decreased immune responses and a germinal cell formation also demonstrated the lack of number of microRNAs overlapped with the mole- correlation between Myc protein and mRNA cular Burkitt lymphoma signature,29,30 including levels.55 The lack of distinct gene expression MIR17HG, which was markedly upregulated, and profiling signatures and better survival of MIR21 (ref. 44) and MIR155HG, which were signifi- MYC-R−/Mychigh GCB-DLBCL and MYC-R+/Mychigh cantly downregulated. Other studies have shown ABC-DLBCL compared with MYC-R+/Mychigh these microRNAs to be regulated by the Myc, GCB-DLBCL may indicate lower Myc activities Bcl-6,STAT3,andNF-κB pathways, and the MIR17HG corresponding to intracellular Myc protein levels; locus was frequently amplified in Burkitt lym- however, small case numbers and/or heterogeneity phoma.45–48 Downregulation of MIR155HG expression among these two Mychigh DLBCL groups could also may contribute to the pathogenesis of MYC trans- be possible causes. location, as miR-155 suppresses activation-induced However, the Myc immunohistochemistry levels cytidine deaminase, which mediates MYC/IGH were similar between MYC-R−/Mychigh GCB-DLBCL translocation.49 These microRNA signatures may also and MYC-R−/Mychigh ABC-DLBCL (although GCB be implicated in defining the gene expression profiling type had slightly lower level of MYC-mRNA) but features of MYC-R+/Mychigh GCB-DLBCL. In lym- their prognosis showed differences. MYC-R+/Mychigh phoma cells, ectopic expression of miR-155 is asso- ABC-DLBCL had similar Myc protein level to that of ciated with downregulation of IGJ, FAS, SMAD3/5, MYC-R−/Mychigh ABC-DLBCL but only the latter and BACH1,aswellasHLAgenes.45 IGJ, FAS,and showed typical Myc gene expression profiling genes involved in BMP/SMAD pathways (such as signatures (Figure 2c). Therefore, Myc-associated SMAD1, BMP3,andBMP7), as well as SMARCA4 molecular mechanisms in GCB or ABC subtype of (miR-21 target gene50), were upregulated in MYC-R+ MYC-R+/ − Mychigh cases impacted the prognostic GCB-DLBCL (Tables 3 and 5). miR-155 target gene and biological effect of Myc. GCB and ABC subtypes BCL2 showed increased Bcl-2 protein expression of Mychigh DLBCL had difference in frequencies of in ABC-DLBCL (Figure 3c). Phosphorylated SMAD TP53 mutation, MYC translocation, BCL2 transloca- proteins have roles in BMP-induced cell growth tion, and Bcl-2 expression, as well as cell-of-origin inhibition (this inhibition can be overcame via MYC biomarkers (Table 1 and Figure 3). MYC-R+/Mychigh

Modern Pathology (2015) 28, 1555–1573 MYC-associated biology and prognosis in DLBCL ZY Xu-Monette et al 1569

Table 6 Summary of frequencies and prognostic significance of MYC-R in DLBCL in the literature and reported by the current study

DLBCL MYC-R+ References cohort frequency Significant prognostic value van Imhoff et al.18 N = 59 15% Nonsignificant trends toward inferior survival Savage et al.12 N=137 8.8% Poorer OS and PFS Oberman et al.14 N = 220 4% Poorer survival Tibiletti et al.19 N = 74 15.8% No prognostic significance Barrans et al.11 N = 245 14% Poorer OS Tapia et al.7 N = 45 20% Akyurek et al.56 N = 239 6% Poorer OS and trend of poorer PFS (P = 0.09) in GCB Kluk et al.5 N =56 9% Green et al.6 N = 219 15% 65% of Mychigh cases Green et al.25 N = 189 11% Poorer OS Johnson et al.22 N = 290 11.7% Inferior OS and PFS when concurrent with Bcl-2high Horn et al.13 N = 407 8.8% Poorer EFS and OS Valera et al.26 N = 176 7% Poorer OS and PFS Aukema et al.16 N = 863 19.5% Tzankov et al.15 N = 432 9% Poorer disease-specific survival Wang et al.8 N = 135 24% Horn et al.10 N = 111 18% Horn et al.27 N = 103 14% Nonsignificant trend towards poorer OS (P = 0.082) Current study N = 344 DLBCL 11.6% Poorer PFS N = 166 GCB 16.3% Poorer OS and PFS N = 177 ABC 7.3% No prognostic significance N = 107 Mychigh cases 24.3% Poorer PFS N = 175* N = 233 Myclow cases 5.6% No prognostic significance N = 360* (a nonsignificant trend of better OS: P = 0.25) N =42 Mychigh GCB 45.2% Poorer OS and PFS N = 76* N =65 Mychigh ABC 10.8% No prognostic significance N = 98* N =60 Mychigh/Bcl-2high DPL 23.3% GCB subtype of MYC-R+ Mychigh/Bcl-2high DPL (16.7%) N = 98* had significantly poorer OS and PFS among Mychigh/Bcl-2high DPL Occurrence of GCB vs ABC subtype of MYC-R+: 2.1; Occurrence of GCB vs ABC subtype of MYC-R+/Mychigh: 2.7; Occurrence of GCB vs ABC subtype of MYC-R+/Mychigh/Bcl-2high: 2.5 Prognosis of GCB vs ABC subtype of MYC-R+: trends towards poorer OS and PFS (P = 0.11); Prognosis of GCB vs ABC subtype of MYC-R+/Mychigh: no significant difference (slightly unfavorable trends); Prognosis of GCB vs ABC subtype of MYC-R+/Mychigh/Bcl-2high: unfavorable trends (for PFS, P = 0.098)

Abbreviations: ABC, activated-B-cell like; Bcl-2high, high Bcl-2 protein expression; DLBCL, diffuse large B-cell lymphoma; DPL, double-positive lymphoma; EFS, event-free survival; GCB, germinal center B-cell like; Mychigh, high Myc protein expression; Myclow, low Myc protein expression; MYC-R+, MYC rearrangement positive; MYC-R¯, MYC rearrangement negative; OS, overall survival; PFS, progression-free survival. Note: Case numbers marked by * are the total Mychigh or Myclow case numbers (with or without MYC-R status determined). and MYC-R−/Mychigh DLBCL had significantly of MYC-R+ DLBCL with Myc overexpression was different levels of MDM2, the cell-of-origin markers, associated with significantly poorer survival, likely and CD30 (in GCB-DLBCL) and pAKT (in contributed by significantly higher Myc protein ABC-DCLBL) (Figure 4), as well as significantly levels as well as associated tumor biology. In different gene expression profiling signatures at the ABC-DLBCL, Myc overexpression associated with mRNA level (Figure 5i). Recently, studies have Bcl-2 overexpression was a significantly adverse posited a ‘c-Myc function rule,’ in which c-Myc is biomarker independent of MYC translocation. Our a ‘universal amplifier’ of active (expressed) genes results suggest that fluorescence in situ hybridization in lymphocytes.33,34,57 analysis for MYC rearrangements and immunohis- In summary, both MYC rearrangement and Myc tochemistry evaluation for Myc and Bcl-2 expression overexpression have advantage and limitations as a are both needed to determine the prognosis in single biomarker in DLBCL, and their prognostic subsets of patients.57 Insights gained into the tumor importance is significantly different in GCB- vs biology associated with MYC abnormalities are ABC-DLBCL. GCB subtype (opposite to the general important for understanding the functional role association of ABC-DLBCL with poorer survival) of MYC in lymphomagenesis and chemoresistance,

Modern Pathology (2015) 28, 1555–1573 MYC-associated biology and prognosis in DLBCL 1570 ZY Xu-Monette et al

Table 7 Summary of frequencies and prognostic significance of Myc overexpression (Mychigh) and MYC mRNA levels in DLBCL in the literature and reported by the current study

Myc overexpression

DLBCL Cutoff for References cohort Mychigh (%) Mychigh frequency Significant prognostic value

Kluk et al.5 N =77 450 19.5% Poorer OS Green et al.6 N = 205 ≥ 70 17% Green et al.25 N = 193 ≥ 40 Poorer OS and PFS when concurrent with Bcl-2high Johnson et al.22 N = 307 ≥ 40 33% Inferior OS and PFS when concurrent with Bcl-2high Hu et al.24 N = 466 ≥ 40 64% Concurrent Mychigh/Bcl-2high correlated with poorer OS and PFS Horn et al.13 N = 283 440 31.8% Poorer OS and PFS Valera et al.26 N = 168 10 48% Inferior OS and PFS 40 13% Perry et al.23 N = 106 450 35% Poorer OS and EFS Horn et al.10 N =39 ≥ 80 77–85% of MYC-R+ cases; 19–46% of MYC-R− cases Horn et al.27 N =92 ≥ 30 49% Nonsignificant trends toward poorer OS (P = 0.08) and poorer PFS (P = 0.091) Current study N = 535 ≥ 70 32.7% Poorer OS and PFS N = 272 ≥ 70 27.9% in GCB Poorer OS and PFS N = 259 ≥ 70 37.8% in ABC Poorer OS and PFS N =40 ≥ 70 67% of MYC-R+ cases Poorer OS and PFS N = 304 ≥ 70 26.9% of MYC-R¯ cases Poorer OS and PFS N =26 ≥ 70 73% of MYC-R+ GCB Trends toward poorer OS (P = 0.07) and PFS N = 136 ≥ 70 16.9% of MYC-R− GCB Nonsignificant trends toward poorer OS (P = 0.40) and poorer PFS (P = 0.48) N =13 ≥ 70 54% of MYC-R+ ABC Trends toward poorer OS and PFS (P = 0.07) N = 164 ≥70 35.4% of MYC-R− ABC Poorer OS and PFS Occurrence of ABC vs GCB subtype of Mychigh: 1.3 (in the overall DLBCL cohort, case numbers of ABC vs GCB subtype: 0.95); Occurrence of ABC vs GCB subtype of Mychigh/Bcl-2high: 1.8 Percentage of MYC-R−/Mychigh ABC-DLBCL among all Mychigh: 54.2%; Percentage of MYC-R+/Mychigh GCB-DLBCL among all Mychigh: 17.8% Prognosis of ABC vs GCB subtype of Mychigh: no significant difference (slightly unfavorable trends); Prognosis of ABC vs GCB subtype of Mychigh/Bcl-2high: no significant difference; MYC-R−/Mychigh ABC vs MYC-R−/Mychigh GCB: trend of poorer OS (for both OS and PFS: P = 0.14); MYC-R+/Mychigh ABC-DLBCL vs MYC-R−/Mychigh GCB-DLBCL: nonsignificantly poorer OS (P = 0.35) and PFS (P = 0.25); MYC-R+/Mychigh GCB-DLBCL vs MYC-R−/Mychigh ABC-DLBCL: a trend towards poorer PFS (P = 0.058)

MYC mRNA levels (3 groups: low, intermediate, and high MYC-mRNA) References Frequency of MYC-mRNAhigh Significant prognostic value

Current study N = 471 15.7% of DLBCL Poorer OS and PFS N = 241 16.6% in GCB Poorer OS and PFS N = 228 14.9% in ABC Poorer OS and a trend towards poorer PFS (P = 0.069) N = 33 55% of MYC-R+ cases Poorer OS and a trend towards poorer PFS (P = 0.066) N = 265 12.1% of MYC-R¯ cases Trend towards poorer OS (P = 0.06) Prognosis of ABC vs GCB subtype of MYC-mRNAhigh: no significant difference (slightly unfavorable trends)

Abbreviations: ABC, activated B-cell like; Bcl-2high, high Bcl-2 protein expression; DLBCL, diffuse large B-cell lymphoma; EFS, event-free survival; GCB, germinal center B-cell like; Mychigh, high Myc protein expression; Myclow, low Myc protein expression; MYC-R−, MYC rearrangement– negative; MYC-R+, MYC rearrangement positive; OS, overall survival; PFS, progression-free survival.

and help identify oncogenic targets for therapeutic (R01CA138688, R01CA187415 and 1RC1CA146299 intervention (Table 7). to YL and KHY). ZYXM is the recipient of the Harold C and Mary L Daily Endowment Fellowships and Acknowledgments Shannon Timmins Fellowship for Leukemia Research Award. GM is supported by a grant from This study was supported by the National the Michael and Susan Dell Foundation. KHY is Cancer Institute/National Institutes of Health supported by The University of Texas MD Anderson

Modern Pathology (2015) 28, 1555–1573 MYC-associated biology and prognosis in DLBCL ZY Xu-Monette et al 1571

Cancer Center Lymphoma Moonshot Program, Insti- MYC gene status in aggressive B cell lymphomas. tutional Research and Development Fund, an Insti- Histopathology 2011;59:672–678. tutional Research Grant Award, an MD Anderson 8 Wang XJ, Medeiros LJ, Lin P et al. MYC cytogenetic Cancer Center Lymphoma Specialized Programs on status correlates with expression and has prognostic Research Excellence (SPORE) Research Develop- significance in patients with MYC/BCL2 protein double-positive diffuse large B-cell lymphoma. Am J ment Program Award, an MD Anderson Cancer Surg Pathol 2015;39:1250–1258. Center Myeloma SPORE Research Development 9 Cheah CY, Oki Y, Westin JR et al. A clinician's guide to Program Award, a Gundersen Lutheran Medical double hit lymphomas. Br J Haematol 2015;168:784–795. Foundation Award, and partially supported by the 10 Horn H, Staiger AM, Vohringer M et al. Diffuse large National Cancer Institute/National Institutes of B-cell lymphomas of immunoblastic type are a major Health (P50CA136411 and P50CA142509), and by reservoir for MYC-IGH translocations. Am J Surg Pathol MD Anderson’s Cancer Center Support Grant 2015;39:61–66. CA016672. 11 Barrans S, Crouch S, Smith A et al. Rearrangement of MYC is associated with poor prognosis in patients with diffuse large B-cell lymphoma treated in the era of rituximab. J Clin Oncol 2010;28:3360–3365. Author contributions 12 Savage KJ, Johnson NA, Ben-Neriah S et al. MYC gene Conception and design: ZYX-M, KHY; research rearrangements are associated with a poor prognosis in performance: ZYX-M, KHY; provision of study diffuse large B-cell lymphoma patients treated with R-CHOP chemotherapy. Blood 2009;114:3533–3537. materials, key reagents and technology: ZYX-M, 13 Horn H, Ziepert M, Becher C et al. MYC status in BSD, XW, MT, GCM, AT, YX, LZ, CV, KD, LY, AC, concert with BCL2 and BCL6 expression predicts AO, YZ, GB, KLR, EDH, WWLC, JHvK, MP, AJMF, outcome in diffuse large B-cell lymphoma. Blood MBM, BMP, XZ, JNW, MAP, TJM, RNM, YL, LJM, 2013;121:2253–2263. KHY; collection and assembly of data under 14 Obermann EC, Csato M, Dirnhofer S et al. Aberrations approved IRB and MTA: ZYX-M, BSD, XW, AT, of the MYC gene in unselected cases of diffuse YX, CV, KD, AC, AO, YZ, GB, KLR, EDH, WWLC, large B-cell lymphoma are rare and unpredictable by morphological or immunohistochemical assessment. JHvK, MP, AJMF, MBM, BMP, XZ, JNW, MAP, KHY; – data analysis and interpretation: ZYX-M, KHY; J Clin Pathol 2009;62:754 756. manuscript writing: ZYX-M, LJM, KHY; final 15 Tzankov A, Xu-Monette ZY, Gerhard M et al. Rearran- gements of MYC gene facilitate risk stratification in approval of manuscript: all authors. diffuse large B-cell lymphoma patients treated with rituximab-CHOP. Mod Pathol 2014;27:958–971. 16 Aukema SM, Kreuz M, Kohler CW et al. Biological Disclosure/conflict of interest characterization of adult MYC-translocation-positive mature B-cell lymphomas other than molecular Burkitt KHY also receives research support from Roche lymphoma. Haematologica 2014;99:726–735. Molecular System, Gilead Sciences Pharmaceutical, 17 Bertrand P, Bastard C, Maingonnat C et al. Mapping of Seattle Genetics, Dai Sanyo Pharmaceutical, Adap- MYC breakpoints in 8q24 rearrangements involving tive Biotechnology, and HTG Molecular Diagnostics. non-immunoglobulin partners in B-cell lymphomas. Leukemia 2007;21:515–523. 18 van Imhoff GW, Boerma EJ, van der Holt B et al. References Prognostic impact of germinal center-associated proteins and chromosomal breakpoints in poor-risk diffuse large 1 Felsher DW. MYC inactivation elicits oncogene B-cell lymphoma. J Clin Oncol 2006;24:4135–4142. addiction through both tumor cell-intrinsic and host- 19 Tibiletti MG, Martin V, Bernasconi B et al. BCL2, BCL6, dependent mechanisms. Genes Cancer 2010;1:597–604. MYC, MALT 1, and BCL10 rearrangements in nodal 2 Delmore JE, Issa GC, Lemieux ME et al. BET bromodo- diffuse large B-cell lymphomas: a multicenter evalua- main inhibition as a therapeutic strategy to target tion of a new set of fluorescent in situ hybridization c-Myc. Cell 2011;146:904–917. probes and correlation with clinical outcome. Hum 3 Chesi M, Matthews GM, Garbitt VM et al. Drug Pathol 2009;40:645–652. response in a genetically engineered mouse model of 20 Johnson NA, Savage KJ, Ludkovski O et al. Lymphomas multiple myeloma is predictive of clinical efficacy. with concurrent BCL2 and MYC translocations: the Blood 2012;120:376–385. critical factors associated with survival. Blood 4 Ott G, Rosenwald A, Campo E. Understanding MYC- 2009;114:2273–2279. driven aggressive B-cell lymphomas: pathogenesis and 21 Visco C, Tzankov A, Xu-Monette ZY et al. Patients classification. Blood 2013;122:3884–3891. with diffuse large B-cell lymphoma of germinal center 5 Kluk MJ, Chapuy B, Sinha P et al. Immunohistochem- origin with BCL2 translocations have poor outcome, ical detection of MYC-driven diffuse large B-cell irrespective of MYC status: a report from an Interna- lymphomas. PLoS One 2012;7:e33813. tional DLBCL rituximab-CHOP Consortium 6 Green TM, Nielsen O, de Stricker K et al. High levels of Program Study. Haematologica 2013;98:255–263. nuclear MYC protein predict the presence of MYC 22 Johnson NA, Slack GW, Savage KJ et al. Concurrent rearrangement in diffuse large B-cell lymphoma. Am J expression of MYC and BCL2 in diffuse large B-cell Surg Pathol 2012;36:612–619. lymphoma treated with rituximab plus cyclophospha- 7 Tapia G, Lopez R, Munoz-Marmol AM et al. Immuno- mide, doxorubicin, vincristine, and prednisone. J Clin histochemical detection of MYC protein correlates with Oncol 2012;30:3452–3459.

Modern Pathology (2015) 28, 1555–1573 MYC-associated biology and prognosis in DLBCL 1572 ZY Xu-Monette et al

23 Perry AM, Alvarado-Bernal Y, Laurini JA et al. MYC B-cell lymphoma with favorable prognosis and distinct and BCL2 protein expression predicts survival in gene expression signature: a report from the Interna- patients with diffuse large B-cell lymphoma treated tional DLBCL Rituximab-CHOP Consortium Program with rituximab. Br J Haematol 2014;165:382–391. Study. Blood 2013;121:2715–2724. 24 Hu S, Xu-Monette ZY, Tzankov A et al. MYC/BCL2 39 Ok CY, Xu-Monette ZY, Li L et al. Evaluation of protein coexpression contributes to the inferior survi- NF-kappaB subunit expression and signaling pathway val of activated B-cell subtype of diffuse large B-cell activation demonstrates that p52 expression confers lymphoma and demonstrates high-risk gene expression better outcome in germinal center B-cell-like diffuse signatures: a report from The International DLBCL large B-cell lymphoma in association with CD30 and Rituximab-CHOP Consortium Program. Blood BCL2 functions. Mod Pathol 2015;28:1202–1213. 2013;121:4021–4031. 40 Chen J, Xu-Monette ZY, Deng L et al. Dysregulated 25 Green TM, Young KH, Visco C et al. Immunohisto- CXCR4 expression promotes lymphoma cell survival chemical double-hit score is a strong predictor of and independently predicts disease progression in outcome in patients with diffuse large B-cell lymphoma germinal center B-cell-like diffuse large B-cell lym- treated with rituximab plus cyclophosphamide, doxo- phoma. Oncotarget 2015;6:5597–5614. rubicin, vincristine, and prednisone. J Clin Oncol 41 Li L, Xu-Monette ZY, Ok CY et al. Prognostic Impact of 2012;30:3460–3467. c-Rel nuclear expression and REL amplification and 26 Valera A, Lopez-Guillermo A, Cardesa-Salzmann T crosstalk between c-Rel and the p53 pathway in diffuse et al. MYC protein expression and genetic alterations large B-cell lymphoma. Oncotarget; advance online have prognostic impact in patients with diffuse large publication, 30 June 2015 [e-pub ahead of print]. B-cell lymphoma treated with immunochemotherapy. 42 Liu Z, Xu-Monette ZY, Cao X et al. Prognostic and Haematologica 2013;98:1554–1562. biological significance of survivin expression in patients 27 Horn H, Ziepert M, Wartenberg M et al. Different with diffuse large B-cell lymphoma treated with rituximab- biological risk factors in young poor-prognosis and CHOP therapy. Mod Pathol; advance online publication, 7 elderly patients with diffuse large B-cell lymphoma. August 2015 [e-pub ahead of print]. Leukemia 2015;29:1564–1570. 43 Huang C, Geng H, Boss I et al. Cooperative transcrip- 28 Pfreundschuh M. Growing importance of MYC/BCL2 tional repression by BCL6 and BACH2 in germinal immunohistochemistry in diffuse large B-cell lympho- center B-cell differentiation. Blood 2014;123: mas. J Clin Oncol 2012;30:3433–3435. 1012–1020. 29 Hummel M, Bentink S, Berger H et al. A biologic 44 Lu TX, Hartner J, Lim EJ et al. MicroRNA-21 limits definition of Burkitt's lymphoma from transcriptional in vivo immune response-mediated activation of the and genomic profiling. N Engl J Med 2006;354: IL-12/IFN-gamma pathway, Th1 polarization, and the 2419–2430. severity of delayed-type hypersensitivity. J Immunol 30 Dave SS, Fu K, Wright GW et al. Molecular diagnosis of 2011;187:3362–3373. Burkitt's lymphoma. N Engl J Med 2006;354:2431–2442. 45 Iqbal J, Shen Y, Huang X et al. Global microRNA 31 Thieblemont C, Briere J. MYC, BCL2, BCL6 in DLBCL: expression profiling uncovers molecular markers for impact for clinics in the future? Blood 2013;121:2165–2166. classification and prognosis in aggressive B-cell lym- 32 Albihn A, Johnsen JI, Henriksson MA. MYC in onco- phoma. Blood 2015;125:1137–1145. genesis and as a target for cancer therapies. Adv Cancer 46 Di Lisio L, Sanchez-Beato M, Gomez-Lopez G et al. Res 2010;107:163–224. MicroRNA signatures in B-cell lymphomas. Blood 33 Lin CY, Loven J, Rahl PB et al. Transcriptional Cancer J 2012;2:e57. amplification in tumor cells with elevated c-Myc. Cell 47 Basso K, Schneider C, Shen Q et al. BCL6 positively 2012;151:56–67. regulates AID and germinal center gene expression 34 Nie Z, Hu G, Wei G et al. c-Myc is a universal amplifier via repression of miR-155. J Exp Med 2012;209: of expressed genes in lymphocytes and embryonic 2455–2465. stem cells. Cell 2012;151:68–79. 48 Testoni M, Zucca E, Young KH et al. Genetic lesions 35 Visco C, Li Y, Xu-Monette ZY et al. Comprehensive in diffuse large B-cell lymphomas. Ann Oncol 2015; gene expression profiling and immunohistochemical 1069–1080. studies support application of immunophenotypic 49 Dorsett Y, McBride KM, Jankovic M et al. algorithm for molecular subtype classification in MicroRNA-155 suppresses activation-induced cytidine diffuse large B-cell lymphoma: a report from the deaminase-mediated Myc-Igh translocation. Immunity International DLBCL Rituximab-CHOP Consortium 2008;28:630–638. Program Study. Leukemia 2012;26:2103–2113. 50 Schramedei K, Morbt N, Pfeifer G et al. MicroRNA-21 36 Xu-Monette ZY, Wu L, Visco C et al. Mutational targets tumor suppressor genes ANP32A and profile and prognostic significance of TP53 in SMARCA4. Oncogene 2011;30:2975–2985. diffuse large B-cell lymphoma patients treated with 51 Holien T, Vatsveen TK, Hella H et al. Bone morpho- R-CHOP: report from an International DLBCL genetic proteins induce apoptosis in multiple myeloma Rituximab-CHOP Consortium Program Study. Blood cells by Smad-dependent repression of MYC. Leukemia 2012;120:3986–3996. 2012;26:1073–1080. 37 Xu-Monette ZY, Moller MB, Tzankov A et al. MDM2 52 Huse K, Bakkebo M, Walchli S et al. Role of Smad phenotypic and genotypic profiling, respective to TP53 proteins in resistance to BMP-induced growth inhibi- genetic status, in diffuse large B-cell lymphoma patients tion in B-cell lymphoma. PLoS One 2012;7:e46117. treated with rituximab-CHOP immunochemotherapy: a 53 Malhotra N, Kang J. SMAD regulatory networks con- report from the International DLBCL Rituximab-CHOP struct a balanced immune system. Immunology Consortium Program. Blood 2013;122:2630–2640. 2013;139:1–10. 38 Hu S, Xu-Monette ZY, Balasubramanyam A et al. CD30 54 Wang M, Saha J, Hada M et al. Novel Smad proteins expression defines a novel subgroup of diffuse large localize to IR-induced double-strand breaks: interplay

Modern Pathology (2015) 28, 1555–1573 MYC-associated biology and prognosis in DLBCL ZY Xu-Monette et al 1573

between TGFbeta and ATM pathways. Nucleic Acids in patients with diffuse large B-cell lymphoma treated Res 2013;41:933–942. with cyclophosphamide, doxorubicin, vincristine, 55 Calado DP, Sasaki Y, Godinho SA et al. The cell-cycle and prednisone plus rituximab. Cancer 2012;118: regulator c-Myc is essential for the formation and 4173–4183. maintenance of germinal centers. Nat Immunol 57 Cai Q, Medeiros LJ, Young KH. MYC-driven aggressive 2012;13:1092–1100. B-cell lymphomas: biology, entity, differential diagno- 56 Akyurek N, Uner A, Benekli M et al. Prognostic sis and clinical management. Oncotarget 2015 [e-pub significance of MYC, BCL2, and BCL6 rearrangements ahead of print].

Supplementary Information accompanies the paper on Modern Pathology website (http://www.nature.com/ modpathol)

Modern Pathology (2015) 28, 1555–1573